6-(alpha - 3 - allophanamidophenylacetamido) and 6 - (alpha - 3 - allophanamidothienylacetamido)-penicillanic acids



United States Patent 3,483,188 6-(a 3 ALLOPHANAMIDOPHENYLACETAMIDO) AND 6 (on 3 ALLOPHANAMIDOTHIENYL- ACETAMIDO)-PENICILLANIC ACIDS Donald Neil McGregor, Fayetteville, N.Y., assignor to Bristol-Myers Company, New York, N.Y., a corporation of Delaware N0 Drawing. Filed June 13, 1968, Ser. No. 736,607 Int. Cl. C07d 99/22; A61k 21/00 US. Cl. 260239.1 22 Claims ABSTRACT OF THE DISCLOSURE 6 (D a allophanamidophenylacetamido) and 6 (D 0c allophanamidothienylacetamido) penicillanic acids and the corresponding -methylallophanamido compounds are valuable as antibacterial agents, nutritional supplements in animal feeds, therapeutic agents in poultry and animals, including man, and are especially useful in the treatment of infectious deseases caused by Grampositive and Gram-negative bacteria, most particularly those caused by the Pseudomonas genus. 6 (D a allophanamidophenylacetamido) penicillanic acid, a preferred embodiment of the invention, is prepared by the reaction of 6 (D a amino phenylacetamido) penicillanic acid (ampicillin) with 1 methyl 1 nitrosobiuret.

BACKGROUND OF THE INVENTION Field of the invention The penicillins of the present invention ponsess the usual attributes of such compounds and are particularly useful in the treatment of Pseudomonas infections.

Description of the prior art Heretofore very few penicillins have been found to possess any significant activity against Pseudomonas. The compounds of the present invention exhibit this desirable activity at significantly low concentrations.

a Aminobenzylpenicillins and a aminothenylpenicillins are known, as from US. Patents 2,985,648, 3,140,282, 3,373,156, 3,308,023 and 3,342,677 and British Patents 903,785, 918,169, 978,178, 991,586 and 1,033,257 and for such compounds bearing substituents on the a-amino group from such British Patents as 891,777, 894,457, 1,053,818, 1,051,675, 1,048,907, 1,064,893, 1,066,107, and 1,080,247 and from South Africa 67/2,092. N-substituted 6-ureidopenicillanic acids have been disclosed, for example, in US. Patents 3,118,877, 3,120,512 and 3,180,863 and in Belgian Patent 603,703 and German Patent 1,120,072; 6-ureidopenicillanic acid itself is disclosed in German Patents 1,141,640 and 1,120,072 and French Patent 1,324,918. A few substituted a-ureidomethylpenicillins are disclosed in US. Patent 3,352,851 and British Patent 1,040,166; none have a substituent in the 3-position of the ureido group. British Patent 1,061,335 discloses 6 (D a hydrazinocarbonylamino a phenyl acetamido)penicillanic acid and 6 (D oz benzyloxycarbonylhydrazocarbonylamino-aphenylacetamido)penicillanic acid and, in general, other so-called acyl groups in place of the benzyloxycarbonyl group.

3,483,188 Patented Dec. 9, 1969 "ice Compounds having the D configuration and formula wherein R is hydrogen or (lower)alkyl, R R and R each is hydrogen, nitro, (lower)alkylamino, di(lower) alkylarnino, (lower)alkanoylarnino, amino, hydroXy, (lower)alkanoyloxy, (lower)alkyl comprising straight and branched chain saturated aliphatic groups having from 1 to 6 carbon atoms inclusive, (lower)alkoxy, sulfamyl, chloro, iodo, bromo, fluoro or trifluorornethyl; X is hydrogen or a nontoxic, pharmaceutically acceptable metallic cation such as sodium, potassium calcium, aluminum, and the like, the ammonium cation and substituted amonium cations, e.g., cations of such nontoxic amines as tri(lower)alkylamines, i.e., triethylamine, etc., procaine, dibenzylamine, N benzyl beta phenethylamine, 1 ephenamine, N,N dibenzylethylenediamine, dehydroabietylamine, N,N' bis dehydroabietylethylenediamine, N (lower)alkylpiperidine, e.g., N ethylpiperidine, or other such amines which have been used to form pharmaceutically acceptable nontoxic salts with benzylpenicillin, are prepared by the treatment of the corresponding a amino compound with 1 methyl-1- nitrosobiuret or a 1 methyl 5 (lower)alkyl 1 nitrosobiuret.

In the preferred embodiments of this invention, R R and R are each hydrogen, hydroxy or amino (and preferably R and R are hydrogen) and R is (lower) alkyl and preferably methyl or, preferably, hydrogen, and the u-carbon of the penicillin side chain (to which the allophanamido group is attached) is of the D() configuration.

DETAILED DESCRIPTION Antibacterial agents such as ampicillin (U.S. 2,985,648) have proved highly effective in the past in the therapy of infections due to Gram-positive and Gram-negative bacteria but these compounds have been notably lacking in their ability to effectively control Pseudomonas infections. Carbenicillin (PYOPEN) (U.S. 3,142,673 and 3,282,926) is active against Pseudomonas only in high concentrations and is useful in such infections in man only when given by injection.

It was an object of the present invention to provide compounds useful in the treatment of infections caused by Gram-positive and Gram-negative bacteria, including particularly those caused by Pseudomonas aeruginv'sa.

The compounds of the present invention as defined above under Summary of the Invention? are particularly useful in that they possess antibacterial activity against both Gram-positive and Gram-negative bacteria, and most particularly exhibit activity against Pseudomonats aerugz'nosa infections.

The a-carbon atom of the penicillin side chain (to which the allophanamido group is attached) is an asymmetric carbon atom and the compounds of this invention can therefore exist in two optically active isomeric forms [the D- and L-diasteroisomers], as well as in the DL form which is a mixture of the two optically active forms; all such isomeric forms of the compounds are included within the scope of the present invention but the D(-) form is preferred. Also included within the scope of the present invention are easily hydrolyzed esters which are converted to the free acid form by chemical or enzymatic hydrolysis.

It should be noted in connection with the foregoing consideration of the diastereoisomers of this invention that many isomers other than the two caused by the asymmetric carbon of the side chain are possible due to the presence of asymmetric carbon atoms in the 6-aminopenicillanic acid nucleus. Such additional isomers, however, are not presently significant since -aminopenicillanic acid which is the product of fermentation processes is consistently of one configuration; such 6-aminopenicillanic acid is presently used in the production of the compounds of this invention.

The compounds of the present invention are prepared by the process comprising the consecutive steps of (a) Mixing a compound having the formula or a monoor polyhydrate or salt thereof,

wherein R R and R each is hydrogen, nitro, di(lower)- alkylamino, (lower)alkanoylamino, amino, hydroxy, (lower)alkanoyloxy, (lower)alkyl (comprising straight and branched chain saturated aliphatic groups having from 1 to 6 carbon atoms inclusive), (lower)alkoxy, sulfamyl, chloro, iodo, bromo, fluoro or trifiuoromethyl; with 1- methyl-l-nitrosobiuret or a l-methyl-S-(lower)alkyl-lnitrosobiuret, preferably in a ratio of 1.0 to 1.5 moles of the nitrosobiuret per mole of the pencillin acid,

in the presence of a base, preferably an organic base such as a pyridine, N-methylpiperidine, tri(lower)alkylamine, or the like,

but preferably in the presence of pyridine or triethylamine, preferably in a ratio of one to two moles of amine per mole of the penicillin acid,

and preferably in a solvent such as water, dimethylformamide, dimethylsulfoxide, chloroform, tetrahydrofuran, n-pentane, methylene chloride, but preferably water,

while preferably maintaining the pH in the range of 7 to 10 and preferably about 9.5 at a temperature in the range of about 2( C. to 50 Q, but preferably about C- to 20 (3.,

for a period of time at least thirty minutes, preferably with agitation, to produce a compound having the formula,

wherein R is hydrogen or (lower)alkyl, R R and R each is hydrogen, nitro, (lower)alkylamino, di(lower)- alkylamino, (lower)alkanoylamino, amino, hydroxy, (lower)alkanoyloxy, (lower)alkyl comprising straight and branched chain saturated aliphatic groups having from 1 to 6 carbon atoms inclusive, (lower)alkoxy, sulfamyl, chloro, iodo, bromo, fiuoro or tri-fiuoromethyl; X is hydrogen or a nontoxic, pharmaceutically acceptable metallic cation such as sodium, potassium calcium, aluminum, and the like, the ammonium cation and substituted ammonium cations, e.g., cations of such nontoxic amines as tri(lower)alkylamines, i.e., triethylamine, etc., procaine, dibenzylamine, N-benzyl-beta-phenethylamine, 1- ephenarnine, N-N' dibenzylethylenediamine, dehydroabietylamine, N-N' bisdehydroabietylethylenediamine, N-(lower)alkylpiperidine, e.g., N-ethylpiperidine, or other such amines which have been used to form pharmaceutically acceptable nontoxic salts with 'benzylpenicillin.

The a-aminopenicillanic acids used as starting materials in the present invention are made by various methods known to the art, the most preferred of which is embodied in US. Patent No. 3,140,282 which employs the condensation of an acid chloride hydrochloride with 6-aminopenicillanic acid to produce an a-aminoarylmethylpenicillin as illustrated below:

In the treatment of bacterial infections in man, the compounds of this invention are administered topically, orally and parenterally in accordance with conventional procedures for antibiotic administration in an amount of from about 5 to mg./kg./day and preferably in the range of 15 to 50 mg./kg./day for Pseudomonas infections in divided dosages, e.g., three or four times a day. They are administered in dosage units containing, for example, 125, 250, 500, 1000 and 2000 mg. of active ingredient with suitable physiologically acceptable carriers or excipients. The dosage units can be in the form of liquid preparations such as solutions, dispersions, emulsions or in solid form such as tablets, capsules, etc.

The nitrosobiurets used as starting materials in the present invention are made by various methods known to the art, the most preferred of which is disclosed by Johrp stOn and Opliger, J. Med. Chem. 10, 67.5 (1967).

Illustrative examples of the preparation of penicillins of the present invention follow. In the title of each example the moiety is represented simply by APA-.

These examples are given in illustration of, but not in limitation of, the present invention. All temperatures are in degrees centigrade. Skellysolve B is a petroleum ether fraction of B.P. 60-68 C. consisting essentially of nhexane.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1 Potassium 6 (D-a-allophanamido-phenylacetamido)- penicillanate.To a stirred solution of 23.7 g.'(0.0588 mole) of ampicillin trihydrate and 8.63 ml. (6.24 g., 0.0616 mole) of triethylamine in 120 ml. of water was added 8.8 g. (0.0602 mole) of 1-methyl1-nitrosobiuret in portions over min. After the addition was complete, stirring was continued for 2 hours. The reaction mixture was layered with 200 ml. of ethyl acetate, cooled in ice, and the pH was adjusted to 2.0 with 42% phosphoric acid. The layers were separated, and the aqueous layer was washed again with 200 ml. of ethyl acetate. The combined organic extracts containing the free acid form of the new penicillin were dried over magnesium sulfate, then concentrated under reduced pressure to a foam. This residue was weighed and dissolved in 250 ml. of acetone, then 200 ml. of acetone containing one equivalent of 50% potassium 2-ethylhexanoate in l-butanol was added. The crystals which separated were collected by filtration, washed with acetone, and dried to give 23.2 g. of potassium 6-(Da-allophanimido-phenylacetamido) penicillanate. Recrystallization from water 2 propanol afforded 17.9 g., M.P. 208210 C. The infrared and NMR spectra were consistent with the assigned structure.

Analysis.CalCd. for C18H20N5O6SK'1/2C3H8O'11AZ- H O: C, 44.14; H, 5.13; N, 13.20. Found: C, 44.27; H, 5.09; N, 13.45.

The Minimum Inhibitory Concentration (M.I.C.) of this product in meg/ml. versus a strain of Pseudomonas aeruginosa was determined by overnight incubation at 37 C. in Nutrient Broth by serial two-fold tube dilution and found to be 16 mcg./ml.; in the same experiment the M.I.C. for sodium ampicillin was found to be 125 mcg./ml. and the M.I.C. for the penicillin of the same structure but in the L(+) configuration [potassium 6-L-a-allophanamido-phenylacetamido)penicillanate] was 250 mgm./ml. and an M.I.C. of 500 meg/ml. was found for the penicillin having the D configuration and the struc ture IIIH O=CCH2(|%-NH3 In another experiment, the M.I.C. of sodium ampicillin was again 125 mcg./ml. and that of sodium D-a-ureidobenzylpeuicillin was 63 mcg./ml.

The median curative dose (CD in mgm./kg. against an overwhelmingly lethal challenge with Pseudomonas aeruginosa (strain A9843) was determined by intramuscular injection of this product in mice of the indicated dosage at the time of challenge and again 3, 6, 24, 27 and 30 hours later; thus a dose of 36 mgm./kg. given at each of these six times is reported as 36x6. Deaths were counted for 72 hours after challenge. The CD found for this product was 170 6 mgm./kg.; the CD for sodium ampicillin in the same experiment was 845 x 6 mgm./kg.

Example 2 O J I N S Potassium 6-(D-a-allophanamido-3-thienylacetamido)- penicillanate.-To a stirred solution of 1.53 g. (0.00374 mole) of 6-(D-a-amino-3-thienylacetamido)-penicillanic acid and 0.55 ml. of (0.00393 mole) of triethylamine in 7.5 ml. of Water was added 0.55 g. (0.00376 mole) of l-methyl-l-nitrosobiuret in portions over 10 min. The reaction mixture was warmed gently to initiate the reaction, then was stirred at ambient temperatures for 2.5 hrs. The solution was layered with 50 ml. of ethyl acetate, cooled in ice, and the pH was adjusted to 2.0 with 42% phosphoric acid. The layers were separated, and the aqueous layer was washed with 50 ml. of ethyl acetate. The combined organic extracts containing the acid form of the new penicillin were dried over magnesium sulfate, then concentrated under reduced pressure to a foam. This was weighed and dissolved in 5 ml. of acetone, then 1 equivalent of 50% potassium Z-ethylhexanoate in l-butanol dissolved in 5 m1. of acetone was added. The mixture was stirred for 30 min., then the solid was collected by filtration, washed with acetone, and dried to give 1.0 g. of potassium G-(D-a-allophanamido 3 thienylacetamido)-penicillanate. Crystallization from 2-propanolwater afforded 0.570 g. of the pure penicillin. The structure is supported by the infrared and NMR spectra.

Analysis.Calcd. for C H N O S K2H O: C, 37.27; H, 4.30; N, 13,58. Found: C, 37.64, H, 4.63; N, 13.59.

The Minimum Inhibitory Concentration (M.I.C.) of this product in meg/ml. versus a strain of Pseudomonas aeruginosa was determined by overnight incubation at 37 C. in Nutrient Broth by serial two-fold tube dilution and found to be 16 mcg./ml.; in the same experiment the M.I.C. for sodium ampicillin was found to be meg/ml.

6-(D-a-allophanamido 2 thienylacetamido)-penicillanic acid.To a stirred solution of 1.53 g. (0.00374 mole) of 6-(D-a-amino-2-thienylacetamido)-penicillanic acid and 0.55 ml. (0.00393 mole) of triethylamine in 7.5 ml. of water was added 0.55 g. (0.00376 mole) of l-methyl-l-nitrosobiuret in portions over 10 min. After the addition, the reaction mixture was stirred for 2 hrs., then 50 ml. of ethyl acetate was added and, with ice cooling, the pH was adjusted to 2.0 with 42% phosphoric acid. The layers were separated and the aqueous layer was washed with 50 ml. of ethyl acetate. The combined organic extracts were dried over magnesium sulfate, then concentrated under reduced pressure. The residue was dissolved in about 10 ml. of acetone, cooled, and scratched. The crystals which separated were collected by filtration, washed with cold acetone, and dried, affording 0.96 g. of 6-(D-a-allophanamido-2-thienylacetamido)- penicillanic acid as the acetone solvate. The structure was confirmed by infrared and NMR spectra.

Analysis.Calcd for C16H19N5O6S2'C3H5O: C, H, 5.04; N, 14.02. Found: C, 46.21; H, 5.44; N, 13.87.

The Minimum Inhibitory Concentration (M.I.C.) of this product in meg/ml. versus a strain of Pseudomonas aeruginosa was determined by overnight incubation at 37 C. in Nutrient Broth by serial two-fold tube dilution and found to be 8 mcg./ml.; in the same experiment the M.I.C. for sodium ampicillin was found to be 125 meg/ml.

Example 4 Potassium 6-(D-oc-(5 methylallophanamido)-phenylacetamido)-penicillanate.To a stirred solution of 2.62 g. (0.0065 mole) of ampicillin trihydratc and 1.04 ml. (0.00743 mole) triethylamine in 10 ml. of water was added 1.19 g. (0.00743 mole) of 1,5-dirnethyl-1-nitrosobiuret in portions over 10 min. After the addition, the mixture was stirred for 4 hrs., then 50 ml. of ethyl acetate was added and, with ice cooling, the pH was adjusted to 2. with 42% phosphoric acid. The layers were separated, and the aqueous layer was washed with 20 ml. of ethyl acetate. The combined organic extracts containing the acid form of the new penicillin were dried over magnesium sulfate, then concentrated under reduced pressure to 2.6 g. of a foam. This was dissolved in 10 ml. of acetone, and a solution of 2.1 g. of 50% potassium Z-ethylhexanoate in l-butanol in 10 ml. of acetone was added. The precipitate which separated was collected by filtration, washed with acetone, and dried, yielding 1.25 g. of the penicillin. Crystallization from water-2-propanol afforded 0.84 g. of the purified penicillin, with consistent infrared and NMR spectra.

Analysis.-Calcd. for C19H22N5O6SK'11/2H2O: C, 44.34; H, 4.90; N, 13.61. Found: C, 44.15; H, 5.03; N, 13.85.

The Minimum Inhibitory Concentration (M.I.C.) of this product in mcg./ml. versus a strain of Pseudomonas aeruginosa was determined by overnight incubation at 37 C. in Nutrient Broth by serial two-fold tube dilution and found to be 16 mcg./ml.; in the same experiment the M.I.C. for sodium ampicillin was found to be 125 mcg./rnl.

Example 5 Potassium 6-(D-u-(S-methylallophanamido)-3-thienylacetamido)-penicillanate.-To a stirred solution of 1.53 g. (0.00374 mole) of 6-(D-u-amino-3-thienylacetamido)- penicillanic acid and 0.55 ml. (0.00393 mole) of triethylamine in 7.5 ml. of water was added 0.61 g. (0.0038 mole) of 1,5-dimethy1-l-nitrosobiuret in portions over 10 min. The reaction mixture was stirred for 3 hrs, then ml. of ethyl acetate was added and, with ice cooling, the pH was adjusted to 2.0 with 42% phosphoric acid. The layers were separated, and the aqueous phase was washed with ml. of ethyl acetate. The combined organic extracts containing the acid form of the new penicillin were dried over MgSO then concentrated under reduced pres sure to a foam. This was weighed and dissolved in 10 ml. of acetone, then 1 equivalent of 50% potassium 2- ethylhexanoate in 1-butanol dissolved in 10 ml. of acetone was added. The precipitate was collected by filtration, washed with acetone, and dried to yield 1.1 g. of potassium 6-(D-ot-(S-methylallophanamido) 3 thienylacetamido)penicillanate. Crystallization from water-Z-propanol alforded 0.820 g. of the purified penicillin, with consistent infrared and NMR spectra.

Analysis.Calcd. for C17H20N505S2K'H20: C, H, 4.34; N, 13.69. Found: C, 40.14; H, 4.78; N, 13.42.

The Minimum Inhibitory Concentration (M.I.C.) of this product in mcg./ml. versus a strain of Pueudomonas aeruginosa was determined by overnight incubation at 37 C. in Nutrient Broth by serial two-fold tube dilution and found to be 8 mcg./ml.; in the same experiment the M.I.C. for sodium ampicillin was found to be 125 mcg./ ml.

Example 6 0 ll CHaNHg HzN-C-NH- G-O C2H5 (I) O N=() 1-methylbiuret.A mixture of 20 g. (0.15 mole) of ethyl allophanate (Eastman) and 76 ml. (0.88 mole) of 40% aqueous methylamine (Rohm and Haas) was heated in a pressure bottle at for 3 hrs. The reaction mixture was cooled, then concentrated under reduced pressure to a colorless solid. Recrystallization from 100% ethanol gave 8.25 g. (46%) of l-methylbiuret, M.P. 172.

l-methyl-1-nitrosobiuret.(Caution should be used in handling this material since it may be toxic. There are indications that it slowly decomposes on storage at 20.) To a stirred, ice-cooled solution of 12.5 g. (0.107 mole) of l-methylbiuret in 96 ml. of 5 N HCl is added dropwise over about 20 min. a solution of 13.4 g. (0.194 mole) of sodium nitrite in 22.5 ml. of water. After the addition was complete, 65 ml. of water was added, and stirring with ice cooling was continued for 1 hr. The precipitate was removed by filtration, washed thoroughly with cold water, then dried to give 13.5 g. of the product, M.P. 145. This method is based on that described by Johnston and Oplinger, J. Med. Chem., 10, 675 (1967).

To a stirred solution of 23.7 g. (0.0588 mole) of ampicillin trihydrate and 8.63 ml. (6.24 g., 0.0616 mole) of triethylarnine in ml. of water was added, in portions over 10 min., 8.8 g. (0.0602 mole) of l-methyl-lnitrosobiuret; since it is possible that one of the side products could be diazomethane, the reaction should be run in the hood. Considerable foaming takes place (the reaction should be run in a large vessel because the foaming is sometimes hard to control) and the temperature This gives a starting pH of 9.5. During the course of the reaction, the pH drops to about 6. In subsequent work with amino acids other than ainpicillin, it was found necessary to maintain the pH at about 9.5 for complete reaction-as the pH drops to 5-6, the reaction stops,

rises to about 38. The foaming seems to stop about 10 min. after the addition is complete; stirring was continued for 2 hours after the addition. The clear reaction mixture was layered with about 200 ml. of ethyl acetate, cooled in ice, and the pH was adjusted to 2.0 with 42% phosphoric acid. The layers were separated, and the aqueous layer was washed again with 200 ml. of ethyl acetate. The combined organic extracts were dried over magnesium sulfate, then concentrated under reduced pressure to an almost-colorless foam. This residue was weighed and dissolved in 250 ml. of acetone, then 200 ml. of acetone containing 1 equivalent of 50% potassium 2-ethylhexanoate in l-butanol was added. The crystals of potassium 6-(D-a-allophanamido-phenylacetamido)-penicillanate which separated were collected by filtration, washed with acetone, and dried to give 23.2 g. Recrystallization from water-2-propanol yielded 17.9 g., M.P. 208210 dec.

Example 7 Dant-acetamido-phenylacetic acid.A suspension of 50 g. (0.331 mole) of D (-)-2-phenylglycine in 700 ml. of water was cooled to to C. and 13.2 g. (0.331 mole) of sodium hydroxide was added with stirring to produce a solution. Acetic anhydride (67.5 g., 0.662 mole) was added rapidly in one portion to the vigorously stirred solution which was initially cooled to 0 to 5 C. by means of a salt-ice cooling bath. This was immediately followed by the addition of a solution of 39.7 g. (0.993 mole) of sodium hydroxide in 200 ml. of water in a rapid stream from a dropping funnel. The temperature rose to a maximum of about 25 C. The solution was stirred for an additional fifteen minutes in the cooling bath and then acidified with concentrated hydrochloric acid. The precipitated product was collected by filtration, washed on the filter with water and recrystallized from 1:1 95% ethanol-water; yield 46.0 g. (72%), M.P. 186-188 C., [a] :217.9 (c. 1%-EtOH).

D-a-acetamido 4 nitrophenylacetic acid.D-u-acetamidophenylacetic acid (20 g., 0.104 mole) was slowly added to 50 ml. of concentrated sulfuric acid with cooling as needed to maintain the temperature at 20 to 25 C. The mixture was stirred for about 20 minutes until most of the solid dissolved. Nitric acid (90%, 01:15, 9.7 ml. 0.208 mole) was added dropwise at such a rate to the stirred mixture that the salt-ice cooling bath maintained the temperature in the range 0 to 5 C. The reaction mixture was stirred at -5 to C. for an additional 30 minutes and then poured onto about 300 g. of ice flakes. The white crystalline product was collected by filtration, washed with water and recrystallized three times from 1:1 95% ethanol-water; M.P. 180 182 C. dec., yield 11.5 g. (46.4%). An additional recrystallization from ethyl acetate did not change the melting point; [a] =206.4 (c. 5%-EtOH).

Analysis.Calcd. for C H N O C, 50.42; H, 4.23; N, 11.76. Found: C, 50.14; H, 4.07; N, 11.96.

D-a-acetamido-4-aminophenylacetic acid.A solution of g. (0.063 mole) of D-a-acetamido-4-nitrophenylacetic acid in 250 ml. of 95% ethanol was hydrogenated in the presence of 0.6 g. of 5% palladium on carbon on a Paar hydrogenator at an initial pressure of 50 p.s.i. for 64 minutes. The product had crystallized from the hydrogenation mixture. Approximately 200 ml. of water was added, the mixture warmed to dissolve the product and the catalyst removed by filtration. Chilling the filtrate gave 9.9 g. of product, M.P. 192-195" C. dec. The product was recrystallized four times from 1:1 95% ethanol-water,

10 weight 4.8 g., M.P. 207-209 C. dec., [a] =182.2 (c. 0.5%-1 N HCl).

AnaIysis.Calcd. for C H N O C, 57.71; H, 5.81; N, 13.46. Found: C, 57,61, 57.64; H, 5.67; N, 13.18.

D-a-acetamido-4-iodophenylacetic acid.To a solution of 5.0 g. (0.024 mole) of D-a-acetamido-4-aminophenylacetic acid in ml. of trifluoroacetic acid at 5 to 0 was added slowly 1.8 g. of sodium nitrite. The solution was stirred for 25 minutes. Solid potassium iodide (4.8 g., 0.024 mole) was added at 0 to 5. The temperature of the dark brown mixture was increased to 30 whereupon a vigorous gas evolution occurred. The mixture was maintained at 30 for 45 minutes and then heated at reflux for one-half hour. The trifluoroacetic acid phase was decanted from the dark colored insoluble material. The trifluoroacetic acid was distilled off at reduced pressure. The residue was taken up in 50 ml. of water. After ice cooling there was obtained a precipitate of brown solid. Two recrystallizations from 1:1 ethanol-water gave D-aacetamido-4-iodophenylacetic acid; M.P. 217 dec. with darkening at 202, [u] :173.2 (c. 0.5 95% ethanol).

Analysis.Calcd. for C H INO C, 37.64; H, 3.16; N, 4.39. Found: C, 37.84; H, 3.30; N, 4.34.

The dark colored insoluble material was slurried With water and treated with 1 M sodium thiosulfate to remove the iodine color. The solid was filtered, washed with water and twice recrystallized from 1:1 95% ethanol-water with a carbon treatment giving additional product:

(c. 0.5 95% ethanol).

AnaIysis.Calcd. for C H INO C, 37.64; H, 3.16; N, 4.39. Found: C, 37.70; H, 3.25; N, 4.45.

D-a-amino-4-iodophenylacetic acid.A suspension of 3.7 g. (0.011 mole) of D-a-acetamido-4-iodophenylacetic acid in 15 ml. of 2 N hydrochloric acid plus suflicient dioxane to give a solution at the boiling point was heated at reflux for 1.5 hours. The solvent was distilled off at reduced pressure. The residue was extracted with water, the insoluble material (solid A) being removed by filtration. The filtrate was stripped to dryness at reduced pressure and the solid residue was extracted with water, the insoluble material (solid B) again removed. The filtrate was again evaporated to dryness and the residue extracted with water and the insoluble material (solid C) again removed. The filtrate was stripped to dryness giving solid D as residue The infrared spectra (KBr) showed solids A and B to be amino acid zwitter ion and solid C to be mostly amino acid hydrochloride.

Solid A was hydrolyzed in 2.5 N hydrochloric acid plus dioxane for 1.75 hours. The solution was evaporated to dryness, the residue taken up in water, a small amount of insoluble material removed by filtration, and the filtrate evaporated to dryness leaving solid E. An infrared spectrum (KBr) showed solid E to be a mixture of amino acid hydrochloride and zwitter ion.

Solids D and E were combined in water and the system adjusted to pH 4.5 giving 1.55 g. of D-a-amino-4-iodophenylacetic acid; M.P. 204205 dec., [a] =99.4 (c. 0.5 1 N HCl).

AnaIysis.Calcd. for C H lNO C, 34.68; H, 2.91; N, 5.06. Found: C, 34.63; H, 3.24; N, 4.77.

D-a-amino 4-iodophenylacetic acid.D-a-acetamido phenylacetic acid (Beil. 14, 591) (200 g., 1.036 mole) was added slowly to a solution of 161.6 g. (0.52 mole) of silver sulfate in 1.2 1. of conc. sulfuric acid with cooling as needed to keep the temperature below 30. Finely pulverized iodine (684 g., 2.7 mole) was added in portions during 1.5 hours. The mixture was stirred at room temperature for 1.5 hours longer. The mixture was filtered through a sintered glass filter and the filtrate poured into ca. 3 l. of crushed ice. The solid was filtered, washed with water, and air dried. The material was recrystallized from 650 ml. of 2-propanol (the hot solution was filtered to remove some insoluble material) giving solid A; yield 46.6 g., M.P.

11 175-183 dec. The filtrate was concentrated and stored in the cold overnight giving solid B; yield 132 g., M.P. 160-168". Solids A and B were crude D-ot-aeetamido-4- iodophenylacetic acid.

Solid B was combined with 500 ml. t 2 N hydrochloric acid and refluxed for one hour. The insoluble material (solid C) was removed by filtration and washed with water; yield 100 g., M.P. 180-183.

Solid C was hydrolyzed in 200 ml. of 2 N hydrochloric acid plus enough dioxane to solubilize the material. After 2.25 hours at reflux the solvent was distilled OR at reduced pressure and the residue extracted with 250 ml. of water. The insoluble material (solid D) was removed by filtration. The filtrate was adjusted to pH 4.5 and after cooling in an ice bath the precipitate was filtered, washed with water, and triturated with boiling 95% ethanol giving 10.8 g. of D-a-arnino-4-iodophenylacetic acid.

Solid A was hydrolyzed in 2 N hydrochloric acid plus dioxane for two hours and the solvent distilled off at reduced pressure. The residue was extracted with water. The insoluble material (solid F) was removed by filtration. The filtrate was adjusted to pH 4.5 giving crystalline D-a-amino-4-iodophenylacetic acid; yield 14.4 g.,

Solids D and F were combined and suspended in 350 ml. of water. The suspension was adjusted to pH 4.5 with 20% sodium hydroxide. The solid was filtered, washed with water, air dried, and triturated with 300 ml. of boiling 95% ethanol giving 42.5 g. of D-a-amino-4-iodophenylacetic acid; [a] =99.8 (c. 0.5 1 N HCl).

D a amino 4 iodophenylacetyl chloride hydrochloride.--A suspension of 42.3 g. (0.15 mole) of finely ground D-u-arnino-4-iodophenylacetic acid in 1.5 l. of methylene chloride was gassed at 0 to 5 with anhydrous hydrogen chloride and 40.6 g. (0.195 mole) of phosphorus pentachloride added. The mixture was stirred for two hours at 5. Skelly Solve B (800 ml.) was added to the reaction mixture and the product collected by filtration. The product was washed with Skelly Solve B and dried in vacuo; yield 40.9 g. (82%).

6 (D 0c amino 4 iodophenylacetamido)penicillanic acid.A suspension of 26.5 g. (0.123 mole) of 6-aminopenicillanic acid in 300 ml. of water at 5 was brought into solution by adjusting to pH 7.0-7.2 with 10% sodium hydroxide. Acetone (1360 ml.), which had been cooled to 0 to 5, was added and the solution adjusted to pH 2.5-3.3 with 6 N hydrochloric acid. D-uamino-4-iodophenylacetyl chloride hydrochloride (40.9 g., 0.123 mole) was added gradually at 0 to 5 while 10% sodium hydroxide was added as needed to maintain the mixture at pH 2.02.8. After the addition was complete the mixture was adjusted to pH 2.9 and stirred for five minutes. After adding 3.6 l. of methyl isobutyl ketone (MIBK) the aqueous phase was separated and the MIBK phase extracted with 360 ml. of water. The aqueous phases were combined and adjusted to pH 4.5 with 10% sodium hydroxide causing the product to crystallize. The product was collected by filtration (filtrate retained) and washed with water and acetone (crop A); yield 14.4 g., M.P. 191- 195 dec. The combined aqueous and acetone washings from crop A were concentrated to remove acetone giving crop B; 4.0 g., M.P. 191l95 dec. The aqueous filtrate from crop A was concentrated giving crop C; 11.7 g., M.P. 191-195 dec. The infrared and nuclear magnetic resonance spectra for the three crops were consistent with 6 (D-a-amino-4-iodophenylacetamido)penicillanic acid.

Analysis.Calcd. for C16H18IN304S'3H2OI C, H, 4.57; N, 7.94. Found: C, 36.88; H, 4.19; N, 7.09.

Potassium 6-[D-u-allophanamido-4-iodophenylacetamido]penicillanate.This compound is prepared by substituting an equimolar weight of 6-(D-a-amino-4-iodophenylacetamido)penicillanic acid for the ampicillin in the procedure of Example 1.

Replacement of the l-methyl-l-nitrosobiuret of Examples 1, 2 and 3 with an equimolar weight of l-methyl- S-ethyl-l-nitrosobiuret produces potassium 6-{D-oz-(5- ethylallophanamido) phenylacetamido}penicillanate, potassium 6-{D-a-(S-ethylallophanamido)-3-thienylacetamido}penicillanate and potassium G-{D-u-(S-ethylallophanamido)-2-thienylacetamido}penicillanate, respectively.

Example 10 D-a-acetamido-3-iodophenylacetic acid.To a solution of 50.0 g. (0.24 mole) of D-u-acetamido-3-aminophenylacetic acid in 550 ml. of trifluoroacetic acid at 5 was added 17.0 g. of 97% sodium nitrite gradually during 10 minutes. The solution was stirred at 5 for 25 minutes longer. The diazonium salt solution was added in a steady stream to a vigorously stirred suspension of 79.0 g. of potassiurn iodide and 2.5 g. of iodine in 300 ml. of trifluoroacetic acid initially at 23. During the addition the temperature rose to 2527 and steady gas evolution was noted. The mixture was stirred for 3.5 hours at room temperature until the gas evolution ceased. The reaction mixture was filtered leaving a quantity of dark gummy solid. The filtrate was concentrated to a small volume, water added, and further concentrated. The concentrate containing a solid was treated with dilute sodium thiosulfate and the solid collected by filtration. Recrystallization from 4:1 water ethanol gave 19.5 g. of D-a-acetamido-3- iodophenylacetic acid; M.P. 181-181.5,

(c. 0.5 95% ethanol).

Analysis.-Calcd. for C H INO C, 37.64; H, 3.16; N, 4.39; I, 39.77. Found: C, 37.80; H, 3.07; N, 4.55; I, 39.20.

D-u-amino-3-iodophenylacetic acid.-D-a-acetamido-3- iodophenylacetic acid (10.0 g.) in 45 ml. of 2 N hydrochloric acid plus sufiicient dioxane to give a solution at the boiling point was refluxed for 2.25 hours. The solvent was evaporated to dryness and the residue extracted with water. The insoluble material (solid A) was removed by filtration. The filtrate was evaporated to dryness and the residue in water was adjusted to pH 4.5 with sodium hydroxide giving 1.0 g. of D-a-amino-3-iodophenylacetic acid; M.P. 192195, [a] =81 (c. 0.5 1 N HCl).

The filtrate deposited a second crop of amino acid on storage in the cold; yield 0.6 g., M.P. 203204.5, [a] =-101.4 (c. 0.5 1 N HCl).

Solid A (which was chiefly amide) was hydrolyzed with 45 ml. 2 N hydrochloric acid plus dioxane for two hours at reflux. The residue remaining after evaporation of the solvent was combined with water and again evaporated to 13 dryness, The residue in water was adjusted to pH 4.5 giving 1.9 g. of D-ozamino-3-iodophenylacetic acid; M.P. 196-199", [a] =95 (c. 0.5 1N HCl).

The infrared and nuclear magnetic resonance spectra of the three fractions were consistent with the'desired product.

D-a-amino-3-iodophenylacetyl chloride hydrochloride. A suspension of 2.5 g. (0.009 mole) of D-aamino- 3-iodophenyl-acetic acid in 100 ml. of methylene chloride at to 5 was gassed with anhydrous hydrogen chloride and 2.5 g. (0.012 mole) of phosphorous pentachloride added. After stirring for 24 hours at 0 to 5 an additional 1.2 g. of phosphorus pentachloride was added and stirring continued for a total of 38 hours. The reaction mixture was diluted with Skelly Solve B, the product filtered, washed with Skelly Solve B, and dried in vacuo; yield 1.2 g.

6 (D u amino 3 iodophenylacetamido)penicillanie acid.-A suspension of 0.9 g. of 6-aminopenicillanic acid in 18 ml. of water at 5 was adjusted to pH 7.07.2 with 10% sodium hydroxide giving a solution. Acetone (72 ml.) which had been cooled to 5 was added and the solution adjusted to pH 2.5-3.3 with 6 N hydrochloric acid. D-uamino-3-iodophenylacetyl chloride hydrochloride (1.2 g.) was added slowly while 10% sodium hydroxide was added as needed to maintain the mixture at pH 2.0-2.5. After the addition of the acid chloride was complete the mixture was adjusted to pH 2.9 and stirring continued for five minutes at 0 to 5. Additional water (18 ml.) and methyl isobutyl ketone (MIBK) were added. The aqueous phase Was separated. The MIBK phase was extracted with 35 ml. of water. The combined aqueous solutions were adjusted to pH 4.5 with 10% sodium hydro'xide causing the product to crystallize. Concentration of the filtrate after removal of the first crop gave a second crop of product; yield both crops, 975 mg, MP. 188- 194 dec.

AnaIysis.Calcd. for C1SH18IN304S'3HZOI C, H, 4.57; N, 7.94; I, 23.98. Found: C, 36.35; H, 4.45; N, 7.8,]; I, 24.15.

Potassium 6-{D-t-allophanamido-3-iodophenylacetamido}penicillanate.This compound is produced by substitution of an equimolar weight of 6-(D-a-amino-3-iodophenylacetamido)-penicillanic acid for ampicillin in the procedure of Example 1.

Example 11 The l-methyl-l-isonitrosobiuret of each of Examples 1, 2 and 3 is replaced by an equimolar weight of the compound of the formula wherein R is n-propyl, isopropyl, n-butyl, isobutyl, sec.-

butyl, t-butyl, n-pentyl and n-hexyl, respectively to produce, respectively, the corresponding compounds of the formulae mole) of D-()-2-(p-hydroxyphenyl) glycine in 100 ml. of glacial acetic acid was bubbled in HCl gas at a vigorous rate for about 5 minutes. At first a clear solution resulted and then the hydrochloride salt crystallized out. Next, 4.45 g. (0.033 mole) of sulfuryl chloride (freshly distilled) in 25 ml. of glacial acetic acid was added, with stirring, over a 30 minute period, dropwise, The temperature was 26- 27 'C. throughout the addition. After one hour stirring, 250ml. of dry ether was added slowly and crystallization began. After 15 min. the product was filtered off, washed with-.dry ether and air dried. The 7 g. obtained was dissolved in 50 ml. of 1 N HCl, filtered, and the pH adjusted with cooling to 5 with cone. NH OH. The resulting crystalline product was filtered off after 5 min. standing, washed with two 20 ml. portions of water and 5X with acetone. The vacu um dried material weighted 4.6 g.; dec. pp. 217 C. (sharp). The NMR and IR spectra were consistent with the desired structure. [a] 137.1 (c.=1%, 1 N HCl).

Analysis.Calcd. for C3H3CINO3Z C, 47.76; H, 4.01; Cl, 1,7,.66..Found: C, 47.16; H, 3.92; Cl, 17.96.

Sodium D- )-N- 2-hydroxyl-naphthylmethylene -ozamino-rx-(3-chloro-4 hydroxyphenyl) acetate. To a stirred solution of 8 g. (0.04 mole) of D-( )-2-( 3-ch1oro- 4-hydroxyphenyl)-glycine, 25 ml. H 0, 10 ml. ethanol, and 1.6 g. (0.04 mole) of sodium hydroxide was added, all at once, a warm solution of 7.57 g. (0.044 mole) of 2-hydroxy-l-naphthaldehyde (Aldrich Chemical Company) in 40 ml. of ethanol. The mixture was heated until an initial precipitate redissolved and then was rapidly cooled to about 5 C. and scratched. After cooling one hour in the ice bath the crystalline product was filtered off and air dried. The bright yellow product was recrystallized from 80% ethanol-20% water to give 10.1 g. of vacuum dried product. The infrared and NMR spectra were entirely consistent with the desired structure.

Analysis.Calculated for C H ClNO Na: C, 60.37; H,3.47. Found: C, 60.66; H, 3.72.

6-[D-()-2,2-dimethyl 4 (3 chloro 4 hydroxyphenyl)-5-oxo-2(H)-limidazolidinyl]penicillanic acid. To a stirred and cooled (10 C.) suspension of 3.78 g. (0.01 mole) of sodium D-(-)-N-(2-hydroxy-l-napthylmethylene)-a-amino-a-(3 chloro 4 hydroxyphenyl)- acetate in ml. of acetone, 5 ml. of p-dioxane and 3 drops of pyridine was added 1.08 g. (0.01 mole) of ethyl chloroformate (EKC). The mixture was stirred at -10 C. for 30 minutes and then cooled to 40 C. and filtered to remove the sodium chloride which precipitated. To this filtrate of the mixed anhydride, vigorously stirred at 15 C., was added all at once, a precooled (0 C.) solution of 2.16 g. (0.01 mole) of 6-aminopenicillanic acid, 1.68 g. (0.02 mole) of NaHCO in 50 ml. of water. There was vigorous CO evolution for about 5 minutes. The temperature was kept at or below 10 C. for 20 minutes and then allowed to come to room temperature (22 C.) for over a 30 minute period. To this solution was added 50 ml. of water and the acetone removed under reduced pressure at 20 C. Two 200 ml. ether extracts were taken and discarded. The aqueous layer was then adjusted to pH 2 with 6 N HCl with suflicient acetone added to keep everything in solution. This solution was allowed to stand 30 minutes at 22 C. and then two 300 ml. ether extracts were taken and discarded. The pH was readjusted to 4.7 with 20% NaOH and concentrated under reduced pressure to a volume of 25 ml. at 20 C. A small amount of insoluble material was filtered off and 25 m1. acetone added to the filtrate. The pH was then adjusted to 8.8 with 20% NaOH and the resulting solution to 3 with 40% H PO and two 100 ml. ethyl acetate extracts taken. The combined ethyl acetate extracts were washed once with 20 ml. H 0 and then filtered and concentrated under reduced pressure at 15 C. to a volume of about 20 ml. The crystalline product was filtered off and slurried in 10 ml. of acetone-water (1:1 by volume) for 10 minutes and filtered again.

The yield was 280 mg. of product decomposing at 182 15 C. and having IR and NMR spectra entirely consistent with the proposed structure.

Analysis.Calcd for C H ClN O C, 51.82; H, 5.04. Found: C, 48.39; H, 5.28.

To a stirred suspension of 600 mg. of 6-[D-()-2,2-dimethyl-4-(3-chloro-4-hydroxyphenyl)-5-oxo-1 imidazolidinyl]-penicillanic acid in 5 ml. water is added 20% sodium hydroxide solution until pH 7 is obtained. The pH is maintained at about 7 with occasional addition of 1 N HCl for four hours and then the pH is adjusted to 4.5 with 1 N HCl and maintained at pH 4.5 for another hour.

The crystalline precipitate that forms is separated by filtration, Washed with water and dried in vacuo over P yielding 102 mg. of the product 6-[D-()-ot-aminoa-(3-chloro-4-hydroxyphenyl) acetamido] penicillanic acid. The infrared spectrum is consistent with the proposed structure having a peak at 1600 emf.

Potassium 6-[D-u-allophanamido-3-chloro-4 hydroxyphenylacetamido]-penicillanate.This product is prepared by substituting an equimolar weight of 6-[D-()-ozamino-a-(3-chloro-4 hydroxyphenyl)acetamidoJpenicillanic acid for the ampicillin in the procedure of EX- ample 1.

Example 13 HOQ-oH-ii-APA-K o=c-Ntr-o-Nm dl-Z-(p-methoxyphenyl)-glycine.To a stirred solution of 19.6 g. (0.4 mole) of NaCN in 80 ml. of H 0 was added 23.6 g. (0.450 mole) of NH Cl and 20 ml. of cone. NH OH followed by 54.5 g. (0.4 mole) of anisaldehyde in 160 ml. of methanol and the temperature maintained at 37 C. for two hours. The methanol was then removed in vacuo and the remaining mixture extracted with two 150 ml. portions of methyl isobutyl ketone (MIBK) and combined. The combined MIBK extracts were washed once with 30 ml. of H 0 and then 240 ml. of 6 N HCl added with good mixing and the MIBK was removed in vacuo. The resulting slurry was heated at reflux (now in solution) for two hours. One hundred ml. of H 0 was added to the hot solution and then 8 g. of decolorizing carbon added and after ten minutes at gentle reflux the carbon was filtered 011 and washed with 50 ml. of hot water. The combined filtrates (hot) were stirred and treated with cone. NH OH until pH 5-6 was obtained (pH paper). The slurry was then cooled to 5 C. and after one hour the crystals were filtered off and washed with two 100 m1. portions of water. The damp cake was then slurried in 250 ml. of water and 50% NaOH added slowly until the product dissolved. Two 300 ml. ether extracts were then taken and discarded. The pH was then adjusted to 5.5 with 6 N HCl with cooling. After one hour the product was filtered 01f, washed with 3 X 100 ml. H 0 and air dried. Yield 40 g.; dec. 244 C. with sublimation at 230 C.

dl-2-(p-methoxyphenyl)-N-(chloro acetyl) glycine. To a stirred suspension of 36 g. (0.2 mole) of dl-2-(pmethoxyphenyD-glycine in 500 ml. of H 0 was added 8 g. (0.2 mole of NaOH pellets and when a clear solution was obtained the solution was cooled to 5 C. and with vigorous stirring 68.2 g. (0.4 mole) of chloroacetic anhydride (war-m) was added all at once. Then a solution of 16 g. (0.4 mole) of NaOH in 100 ml. of H 0 was added over a to minute period. More NaOH was added as needed to keep the pH at about 9 for a 1.5 hour period. Next, the pH was adjusted to 2 with 40% H PO The product crystallized immediately and was filtered off, washed with water and recrystallized from ethanol-water to give 38 g. of product melting at 182:

Analysis.-Calcd for C H ClNO C, 51.21; H, 4.69. Found: C, 51.49; H, 4.90.

D-(-)-2-(p-methoxyphenyl) N chloroacetylglycine and L-(+)-2-(p-methoxyphenyl)-glycine.-To 800 ml. of H 0 stirred at 37 C. was added 38 g. (0.148 mole) of dI-Z-(p-methoxyphenyl) N chloro-acetylglycine and NH OH added dropwise until pH 7.8 was obtained. To the resulting solution was added 2 g. of Hog Kidney Acylase (Sigma Chemical Company) and stirring continued at 37 C. (internal) for 21 hours. The solids containing crude L-(+)-2-(p-methoxyphenyl)-glycine were then filtered off and washed with 2X ml. H 0 and the pH of the combined filtrates adjusted to 4-5 with glacial acetic acid. This solution was heated on the steam bath for 30 min. with 5 g. of decolorizing carbon and then filtered. The carbon cake was Washed with 50 ml. of warm water and the combined filtrates cooled and acidified to pH 2 with 40% H PO After one hour cooling at 0 C. the crystalline product was filtered off and washed with cold water (3X) and air dired. The yield was 16 g. D- -2- p-methoxyphenyl -N-chloroacetylglycine and when a second run using 5x the above amounts were used a. yield of 83 g. (87% yield) was obtained. M.P. -171 C.; [m] -193 (c.=1%, ethanol).

Analysis.-Calcd. for C H ClNO C, 51.21; H, 4.69. Found: C, 51.50; H, 4.99.

When the solids containing crude L-(+)-2- (p-methoxyphenyl)-N-chloroacetylglycine are treated with hot 3 N HCl (200 ml.) and carbon followed by filtration and pH adjustment to 5.5 there is obtained 6 g. (first run) of pure L-(-|-)-2-(p-methoxyphenyl)glycine. [a]-, 150.4 (0. 1%, 1 N HCl).

D-()-2-(p-methoxyphenyl)-glycine.-The 16 g. of D- -2-(p-methoxyphenyl)-N-(chloroacetyl) -glycine was refluxed 1.5 hours in 170 ml. of 2 N HCl. The resulting clear solution was filtered and cooled at 5 C. and the pH adjusted to 5.5 with NH OH. The product was then filtered off after cooling 30 min. and washed with 3x 25 ml. of cold water. The dried material D-(-)-2-(pmethoxyphenyl)-glycine weighed 9.5 g. A second run gave 54 g.

Analysis.Calcd. for C H NO C, 59.67; H, 6.13; N, 7.74. Found: C, 59.38; H, 6.16; N, 8.00.

D-()-2-(p-hydroxyphenyl)-glycine.-A mixture of 1.81 g. (0.01 mole) of D-()-2-(p-methoxyphenyl) glycine. ([a.] 149'.9 c.=l%, 1 N HCl) and 10 ml. of 48% HBr was heated at gentle reflux for 2 hours. The resulting solution was concentrated at reduced pressure at 30 C. to a wet solid. A minimum amount of water (20 C.) was added to dissolve the HBr salt and with cooling NH OH was added to pH 5. The resulting thick gel which ppt. was warmed to 50 C. and when solution was nearly obtained a different crystalline form began to ppt. Upon cooling 30 min. at 05 C. there was obtained 990 mg. of cold water washed (3X 1 ml.) and air dried material, D-(--)-2-(p-hydroxyphenyl)- glycine. [u] 161.2 (c.=1%, 1 N HCl) dec. pt. 223 C. A second run using 20X the above amounts gave 24.5 g. of material. [od -153 (c.=l%, 1 N HCl).

A.nalysis.Ca1cd. for C H NO C, 57.49; H, 5.43; N, 8.39. Found: C, 57.41; H, 5.67; N, 8.39.

D-(-)-2-(3,5-dichloro-4-hydroxyphenyl) glycine.- To a stirred suspension of 5.01 g. (0.03 mole) of -D-(-)- 2-(4-hydroxyphenyl)glycine in 100 ml. of glacial acetic acid was bubbled in HCl gas at a vigorous rate for about 5 minutes. At first a clear solution resulted and then the hydrochloride salt crystallized out. Next, 9.0 g. (0.067 mole) of sulfuryl chloride (freshly distilled) in 25 ml. of glacial acetic acid was added, with stirring, over a 30 minute period, dropwise. The temperature was 26- 27 C. throughout the addition. After the sulfuryl chloride addition, the slurry was heated to 70 C. for 30 minutes and then stirred at ambient temperature for two hours. Then 250 ml. of dry ether was added slowly and crystal lization began. After 15 min. the product was filtered off, washed with dry ether and air dried. The 7 g. obtained was dissolved in 100 ml. of 1 N HCl, filtered, and the pH adjusted, with cooling to 5 with cone. NH OH. The resulting crystalline product was filtered off after 5 min. standing, washed with two 20 ml. portions of water and 5X with acetone. The vacuum dried material weighed 4.5 g.; dec. pt. 210 C. (sharp). The NMR and IR spectra were consistent with the desired structure. [a] -l26 (c.=1%, 1 N HCl).

Analysis.-Calcd. for C H Cl NO3: C, 40.78; H, 2.99; CI, 30.04. Found: C, 41.85; H, 3.22; C1, 2780.

Sodium D- -N- (Z-hydroxy-l-naphthylmethylene) 2-amino-2-(3,5-dichloro 4 hydroxyphenyl)-acetate). To a stirred solution of (0.04 mole) of D-()-2-(3,5- dichloro-4-hydroxyphenyl)-glycine 25 ml. H 0, 10 ml. ethanol, and 1.6 g. (0.04 mole) of sodium is added, all at once, a warm solution of 7.57 g. (0.044 mole) of 2-hydroxy-l-naphthalaldehyde (Aldrich Chemical Company) in 40 ml. of 95% ethanol. The mixture is heated until an initial precipitate redissolves and then is rapidly cooled to about 5 C. and scratched. After cooling one hour in the ice bath the crystalline product is filtered off and air dried. The infrared and NMR spectra are entirely consistent with the desired structure.

6 [D 2,2 dimethyl 4 (3,5 dichloro 4- hydroxyphenyl) 5 oxo 1 imidazolidinyl]penicillanic acid.To a stirred and cooled (l C.) suspension of (0.01 mole) of sodium D-()-N-(2-hydroxy-1 napthalmethylene) a amino oz (3,5 dichloro 4 hydroxyphenyl) acetate in 100 ml. of acetone, ml. of p-dioxane and 3 drops of pyridine is added 1.08 g. (0.01 mole) of ethyl chloroformate (EKC). The mixture is stirred at 10 C. for 30 minutes and then cooled to 40 C. and filtered to remove the sodium chloride which precipitated. To this filtrate of the mixed anhydride, vigorously stirred at C. is added, all at once, a precooled (0 C.) solution of 2.16 g. (0.01 mole) of 6-aminopenicillanic acid, 1.68 g. (0.02 mole) of NaHCO in 50 ml. of water. There is vigorous CO evolution for about 5 minutes. The temperature is kept at or below -l0 C. for minutes and then allowed to come to room temperature (22 C.) for over a 30 minute period. To this solution is added 50 ml. of water and the acetone removed under pressure at 20 C. Two ml. ether extracts are taken and discarded. The aqueous layer is then adjusted to pH 2 with 6 N HCl with suflicient acetone added to keep everything in solution. This solution is allowed to stand 30 minutes at 22 C. and then two 300 ml. ether extracts are taken and discarded. The pH is readjusted to 4.7 with 20% NaOH and concentrated under reduced pressure to a volume of 25 ml. at 20 C. A small amount of insoluble material is filtered ofi and 25 ml. acetone added to the filtrate. The pH is then adjusted to 8.8 with 20% NaOH and the resulting solution let stand at 10 C. for 5 hours. The pH is adjusted to 3 with 40% H PO and two 100 ml. ethyl acetate extracts taken. The combined ethyl acetate extracts are washed once with 20 ml. H 0 and then filtered and concentrated under reduced pressure at 15 C. to a volume of about 20 ml. The crystalline product-is filtered off and slurried in 10 mlgof acetone-water (1:1 by volume) for 10 minutes and filtered again.

The product obtained D-()-6-[2,2-dimethyl-4-(3,5- dichloro 4 hydroxyphenyl) 5 oxo 1 imidazolidinyl]penicillanic acid and having IR and NMR spectra entirely consistent with the proposed structure.

6 [D a amino a (3,5 dichloro 4 hydroxyphenyl)-acetamido]penicilla nic acid-To a stirred suspension of 600 mg. of 6-[D-()-2,2-dimethyl-4-(3,5- dichloro 4 hydroxyphenyl) 5 oxo 1 imidazolidinyl]-penicillanic acid in 5 ml. of water is added 20% sodium hydroxide solution until pH 7 is obtained. The pH is maintained at about 7 withoccasional. addition of 1 N HCl for four hours and then the pH is adjusted to 4.5 with 1 N HCl and maintained at pH 4.5 for another hour. The crystalline precipitate that forms is separated by filtration, washed with water and dried in vacuo over P 0 yielding the product 6-[D-(-)-a-amino-u-(3,5-dichloro-4-hydroxyphenyl) acetamido] penicillanic acid. The infrared spectrum is consistent with the proposed structure having a peak at 1600 cmf Potassium 6 {D or allophanamido 3,5 dichloro- 4 hydroxyphenylacetamido}penicillanate.The productis prepared by substituting an equimolar weight of 6-[D- 0c amino a (3,5 dichloro 4 hydroxyphenyl)- acetamido1penicillanic acid for the ampicillin in the procedure of Example 1.

Example 14 Potassium 6 {D a allophanamido 4 hydroxyphenylacetamido}penicillanate.-This product is prepared by substituting for the ampicillin in the procedure of Example 1 an equimolar weight of 6-[D-()-a-amino-a- (4-hydroxyphenyl)acetamido]penicillanic acid which is prepared from D-a-(4-hydroxyphenyl)glycine, as by the methods known in the art and that given above for D-OL- (3,5-dichloro-4-hydroxyphenyl) glycine and D-a-amino-4- iodophenylacetic acid [also called D-u-(4-iodophenyl) glycine].

Potassium 6 {D oz allophanamido 3 hydroxyphenylacetarnido}penicillanate.-This product is prepared by substituting for the ampicillin in the procedure of Example 1 an equimolar weight of 6-[D-()-a-amino-u- (3-hydroxyphenyl)acetamido]penicillanic acid which is prepared from D-a-(3-hydroxyphenyl) glycine, as by the methods known to the art, e.g., British Patent 991,586, and those illustrated above for D-a-(3,5-dichloro-4hydroxyphenyl) glycine and D-ot-(4-iodophenyl) glycine.

D-a-(3-hydroxyphenyl)glycine is prepared by the methods known to the art and also, preferably, by the following procedures:

1) Resolution of DL-u-amino-3-methoxyphenylacetic acid.DL-a-amino-3-methoxyphenylacetic acid [A. H. Neims, D. C. DeLuca, L. Hellerman, Biochemistry, 5 (1), 203 (1966)] was resolved with d-IO-carnphorsulfonic acid in water.

DL-a-amino-3-methoxyphenylacetic acid (33.1 g., 0.182 mole) was added to a solution of 46.4 g. (0.2 mole) of d-IO-camphorsulfonic acid in ml. of water at 50 to 60. The solution was filtered and stored in the cold for 20 hours. The precipitated amino acid d-IO-camphorsulfonate salt was collected by filtration. The salt was re peatedly recrystallized from water until a sample of the amino acid regenerated from it showed no further change in optical rotation. Thus, after three recrystallizations from water, there was obtained 3.7 g. of the d-10-camphorsulfonate salt of D-u-amino-3-methoxyphenylacetic acid; M.P. 184185 dec. The salt (1.4 g.) was dissolved in about 10 ml. of water by warming. The solution was adjusted to pH 5-6 with concentrated ammonium hydroxide. The product was allowed to crystallize first at room temperature and then in an ice bath giving, after filtration and drying in vacuo over phosphorus pentoxide, 0.36 g.

19 of D-a-amino-3-methoxyphenylacetic acid; M.P. 178- 181 dec., [u] =-l29.0 (c. 0.5 1 N HCl). A portion of the amino acid was recrystallized from water and dried in vacuo over phosphorus pentoxide; M.P. ISO-182 dec., [a] =136 (c. 0.08 1 N HCl).

Analysis.Calcd. for C H NO -1/3 H O: C, 57.74; H, 6.28; N, 7.48. Found: C, 57.70; 57.76; H, 6.23; 6.18; N, 7.21.

D-a-amino-3-hydroxyphenylacetic acid hydrobromide monohydrate. D-a-amino-3-methoxyphenylacetic acid (2.9 g., 0.016 mole) and 16 ml. of 48% hydrobromic acid were refluxed for two hours. The volatile materials were removed at reduced pressure. Water (about 15 ml.) was added to the residue and this removed at reduced pressure. This was repeated once. The residue was dried in vacuo to remove all water. The dried residue was recrystallized by dissolving in 2-propanol and adding Skellysolve B to the cloud point. After drying there was obtained 3.0 g. of Da-amino-3-hydroxyphenylacetic acid hydrobromide monohydrate; M.P. 156162 dec., [u] =62 (c. 0.1 water). The infrared and nuclear magnetic resonance spectra were consistent with the desired product.

Analysis.Calcd. for C H NO -HBr-H O: C, 36.10; H, 4.55; N, 5.26. Found: C, 37.03; H, 5.12; N, 5.34.

(2) D-a-acetamido-3-nitrophenylacetic acid.A stirred suspension of 49.8 g. (0.254 mole) of D-a-arnino-3-nitro phenylacetic acid [P. Fries, K. Kjaer, Acta Chimica Scand., 17, 2391 (1963)] in 500 ml. of water was cooled in an ice bath and a solution of 8.36 g. (0.209 mole) of NaOH in 40 ml. of water was added causing most of the solid to dissolve. There was immediately added 42.7 g. (0.418 mole) of acetic anhydride followed by the addition as needed of a solution of 25.1 g. (0.627 mole) of sodium hydroxide to maintain the pH value at about 7. The reaction mixture was stirred in the ice bath for an additional 15 minutes, filtered, and adjusted to pH 1.8 with concentrated hydrochloric acid. The crystalline product was collected by filtration and washed with water; yield 25 g., M.P. 172174 dec. The product was twice recrystallized from 1:1 95% ethanol-water giving, after drying in vacuo over phosphorus pentoxide, 11.8 g. of D-a-acetamido-3-nitrophenylacetic acid, M.P. 183-185 [a] =-179.4 (c. 0.5 95% ethanol). The infrared and nuclear magnetic resonance spectra were consistent with the desired compound.

Analysis.Calcd. for c H,,N,0,: C, 50.42; H, 4.23; N, 11.76. Found: C, 50.56; H, 4.20; N, 11.73.

D-u-acetamido-3-aminophenylacetic acid-A solution of 9 g. (0.0378 mole) of D-a-acetamido-3nitrophenyl acetic acid in 150 ml. of methanol was hydrogenated using 0.6 g. of palladium on carbon at an initial pressure of 50 p.s.i. on a Paar hydrogenation apparatus for 30 minutes. The hydrogenation bottle was cooled with a jet of air to keep the temperature under 40. The catalyst was removed by filtration. Evaporation of the filtrate gave a crystalline product. Two recrystallizations from 1-pro panol gave 3.4 g. of D-rx-acetamido-3-aminophenylacetic acid, M.P. ZOO-201 dec.; [e] =174.4 (c. 0.5 water).

Analysis.-Calcd. for C H N O C, 57.71; H, 5.81; N, 13.46. Found: C, 57.78; H, 5.97; N, 13.35.

D-a-amino-3-hydroxyphenylacetic acid.A solution of 2.1 g. (0.01 mole) of D-a-acetamido-3-aminophenylacetic acid in 35 ml. of trifluoroacetic acid was cooled to 5 and 0.69 g. (0.01 mole) of solid sodium nitrite added. After stirring for minutes at 5 acetic acid (5 ml.) was added. The mixture was stirred at 45 to 50 for one and one-half hours and then heated on the steam bath for one-half hour. The cold reaction mixture was poured onto 30 g. of crushed ice. The volatile materials were distilled at reduced pressure leaving as residue a slightly brown viscous oil. The residue was combined with 30 ml.

this removed under reduced pressure causing the hydrochloride salt of 'the product to crystallize. The residue Was dissolved in a minimum amount of water, adjusted to pH 4.5 with 20% sodium hydroxide, filtered, and stored in the cold giving 0.43 g. of crystalline D-a-amino-3-hydroxyphenylacetic acid, M.P. 204-206 dec. The filtrate was stripped to dryness and a small amount of water added to the crystalline residue giving a 2nd crop (0.46- g.) of the amino acid.

The filtrate from the 2nd crop plus 3 ml. of concent'rated hydrochloric acid were concentrated to dryness. The residue was dissolved in a small amount of water by warming, cooling gave crystalline D-a-amino-3-hydroxyphenylacetic acid hydrochloride monohydrate; yield 0.5 g., M.P. 150-153 dec., [a] =-9l.2 (c. 0.5 water). The infrared and nuclear magnetic resonance spectra were consistent with the desired product.

Analysis.Calcd. for C H NO -HCl-H O: C, 43.35; H, 5.46; N, 6.32. Found: C, 42.7; H, 5.6; N, 6.17; residue, 1.45. Values corrected for 1.45% residue: C, 43.3; H, 5.7; N, 6.26.

The two crops of D-a-amino-3-hydroxyphenylacetic acid were combined, suspended in a small amount of water, 2 ml. of 48% hydrobromic acid added and the filtered solution evaporated to dryness. The residue was twice recrystallized from water giving 150 mg. of D-a-arnino-3- hydroxyphenylacetic acid hydrobromide monohydrate; M.P. 172-175 dec., a =-74 (c. 0.1 water). The infrared and nuclear magnetic resonance spectra were consistent with the assigned structure.

Analysis.-Calcd for C H NO -HBr'H O: C, 36.10; H, 4.55; N, 5.26. Found: C, 36.20; H, 4.62; N, 5.32.

(3) A solution of 38.4 g. (0.1845 mole) of D-a-acetamido-3-aminophenylacetic acid in 600 ml. of trifluoroacetic acid prepared at 15 to 20 was cooled to 5 and 13 g. (0.1845 mole) of 98% sodium nitrite added in portions during a 10-minute period with stirring at 5. After stirring for and additional 25 minutes 90 ml. of

acetic acid was added at 5 to 0. The mixture was heated at 45 to 50 for one and one-half hours (gas evolution), refluxed for one-half hour, cooled, and poured onto 500 g. of ice flakes. The volatile materials were removed at reduced pressure. The residue was refluxed with 400 ml. of 2 N hydrochloric acid for one hour. Concentration to a small volume gave the cr stalline hydrochloride salt. The dried product (27 g.) was recrystallized from wet acetic acid ml. acetic acid plus 7 ml. of water) giving 21 g. of D-a-amino-3-hydroxyphenylacetic acid hydrochloride monohydrate; M.P.

' mama 6 -{D-u-allophanarnido-(3- and 4-acetamido)- aw smf d um hy o de wa added w th st g give ing a solution. Acetic anhydride (67.5 g., 0.662 mole) was added rapidly in one portion to the vigorously stirred solution which was initially cooled to to by means of a salt-ice cooling bath. This was immediately followed by the addition of a solution of 39.7 g. (0.993 mole) of sodium hydroxide in 200 ml. of water in a rapid stream from a dropping funnel. The temperature rose to a maximum of about 25. The solution was stirred for an additional fifteen minutes in the cooling bath and then aciditied with concentrated hydrochloric acid. The precipitated product was collected by filtration, washed on the filter with water and recrystallized from 1:1 95% ethanolwater; yield 46.0 g. (72%), M.P. 186188,

(c.:1, 95 EtOH). This compound is reported in Beil. 14, 591.

D-a-acetamido -4 nitrophenylacetic acid.D-a-acetamidophenylacetic acid (20 g., 0.104 mole) was slowly added to 50 ml. of concentrated sulfuric acid with cooling as needed to maintain the temperature at 20 to 25. The mixture was stirred for about 20 minutes until most of the solid dissolved. Nitric acid (90%, d=1.5, 9.7 ml., 0.208 mole) was added dropwise at such a rate to the stirred mixture that the salt-ice cooling bath maintained the temperature in the range 0 to 5. The reaction mixture was stirred at -5 to for an additional 30 minutes and then poured onto about 300 g. of ice flakes. The white crystalline product was collected by filtration, washed with water and recrystallized three times from 1:1 95% ethanol-water; M.P. 180182 dec., yield 11.5 g. (46.4%). An additional recrystallization from ethyl acetate did not change the melting point;

[a] =206.4 (c.=0.5, 95% EtOH).

Analysis.Calcd for C H N O C, 50.42; H, 4.23; N, 11.76. Found: C, 50.14; H, 4.07; N, 11.96.

D-ot-acetamido-4-aminophenylacetic acid.A solution of g. (0.063 mole) of D-a-acetamido-4-nitrophenylacetic acid in 250 ml. of 95 ethanol was hydrogenated in the presence of 0.6 g. of 5% palladium on carbon on a Paar hydrogenator at an initial pressure of 50 p.s.i. for 64 minutes. The product had crystallized from the hydrogenation mixture. Approximately 200 ml. of water was added, the mixture warmed to dissolve the product and the catalyst removed by filtration. Chilling the filtrate gave 9.9 g. of product, M.P. 192-195 dec. The product was recrystallized four times from 1:1 95% ethanol-water; wt. 4.8 g., M.P. 207209 dec.,

(c.=0.5, 1 N HCl).

Analysis.Calcd for C H N O C, 57.71; H, 5.81; N, 13.46. Found: C, 57.61, 57.64; H, 5.67; N, 13.18.

D-a-amino-4 acetamidophenylacetic acid.D-a-acetamido-4-aminophenylacetic acid (39.2 g., 0.188 mole) in 400 ml. of 2 N hydrochloric acid Was refluxed for 2 hours. The mixture was concentrated to dryness at reduced pressure. Water was added and the solution again concentrated to dryness. This was repeated once. The crystalline residue was slurried with 2-propanol, filtered, and washed additionally with 2-propanol giving, after air drying, 47 g. of the hydrochloride of D-a-amino-4-aminophenylacetic acid.

Ten g. of the hydrochloride in 40 ml. of water was adjusted to pH 4.8 with sodium hydroxide. Crystalline D-a-amino-4-aminophenylacetic acid separated. To the solution obtained by adding 160 ml. additional of water was added 10 ml. of thioacetic acid. The mixture was stirred for 17 hours at 24 under a nitrogen atmosphere. The reaction mixture, containing a quantity of crystalline product, was concentrated to one-half of its initial volume giving 4.5 g. of product. The crude product was suspended in water, the suspension adjusted to pH 4.6 with 20% NaOH, heated to 95, carbon treated, and

the product allowed to crystallize in the cold overnight. The resulting gelatinous mass was broken up by warming. The solid was removed by filtration; wt. 0.2 g., M.P. 203- 206 dec. The filtrate was diluted with an equal volume of ethanol giving 1.4 g. of D-u-amino-4-acetamidophenylacetic acid; M.P. 214-215 dec., [a] =133.4 (c.=0.5, 1N HCl).

Analysis. Calcd for C H N O C, 57.71 H, 5.814; N, 13.46. Found: C, 56.80, 56.72; H, 5.84, 5.89; N, 13.62; H O, 1.32. Found values corrected for 1.32% water: C, 57.52;H, 5.71;N, 13.80.

(B) D a-arnino-3-aminophenylacetic acid.A solution of 9.8 g. (0.05 mole) of D-a-amino-3-nitrophenylacetic acid [P. Friis and A. Kjaer, Acta Chimica Scand. 17, 2391 (1963); British patent specification, 1,033,257] in 200 ml. of water was prepared by adjusting the mixture to pH 9.3 with concentrated ammonium hydroxide. The solution was hydrogenated for 1 hour in the presence of 0.4 g. of 5% palladium on carbon on a Paar hydrogenation apparatus at an initial pressure of 50 p.s.i. The vessel was cooled as needed to keep the temperature from going above 30. After 1 hour an additional 0.4 g. of catalyst was added and hydrogenation continued for 1 hour longer. Three additional runs were made hydrogenating a total of 39.4 g. of nitro compound. Addition of the second amount of catalyst was omitted in the additional runs and a hydrogenation time of about 1 hour was used. Each run was filtered to remove catalyst, the filtrates pooled and concentrated to a small volume until crystallization of the product started. The concentrate was diluted with about five volumes of 95 ethanol, the mixture stored overnight in the cold and the product filtered and washed further by slurring with 95% ethanol. After drying in a vacuum oven for 3 hours at 40 and then in vacuo over phosphorus pentoxide for 64 hours there was obtained 25.7 g. of D-u-amino-3-amino-phenylacetic acid; M.P. 188-191, [a] =139.0 (c.=1 N HCl).

The preparation of this compound has been described by P. Friis and A. Kjaer, Acta Chimica Scand. 17, 2391 (1963).

D-a-amino 3 acetamidophenylacetic acid.A mixture of 5 g. (0.0301 mole) of D-a-amino-3-aminophenylacetic acid and 5 ml. of thioacetic acid in ml. of water was stirred for 16 hours under a nitrogen atmosphere. The mixture was heated on a steam bath for onehalf hour and then concentrated at reduced pressure to a small volume. On cooling the concentrate the product started to crystallize. The concentrate was diluted with 95 ethanol and, after chilling in an ice bath, the prodnot was filtered and washed with 95% ethanol; wt. 1.8 g. The filtrate was further diluted and washed with 95 ethanol giving an additional 2.3 g. of product. The two crops of product were combined, dissolved in a small amount of water by warming, the solution concentrated slightly and diluted with a large volume of 95% ethanol. The initial crop of solid was removed by filtration and the filtrate stored in the cold for 16 hours giving, after drying at 65 for 3 hours in vacuo over phosphorus pentoxide, 0.90 g. of product, M.P. -187 dec. The product was twice recrystallized from 1:1 95 ethanolwater; wt. 0.36 g., M.P. 186187 dec., [a] =120 (c.=0.5, 1 N HCl).

Analysis.--Calcd. for C10H12N203: C, H, N, 13.5. Found: C, 47.29; H, 6.79; N, 11.21; H O, 18.3. Found values corrected for 18.3% water: C, 57.9; H, 5.83; N, 13.7.

Potassium 6-{D-u-allophanamido 3 aminophenylacetamido}penicillanate.-This product is prepared by substituting for the ampicillin in the procedure of Example 1 an equimolar weight of 6-[D-a-amino-3-amin0- phenylacetamindo]-penicillanic acid which is prepared in turn either by the procedure of British Patent 1,033,257 or from 2-(3-amino-phenyl) glycine by the acylation procedure of US. Patent 3,140,282.

Example 18 When in the procedure of Example 1 the D-u-a-minobenzyl-penicillin used therein is replaced with 59 mtnoles of:

D-a-amino-p-sulfamylbenzylpenicillin, D-a-amino-p-chlorobenzylpenicillin, D-u-arnin-p-methoxybenzylpenicillin, D-a-arnino-p-diethylaminobenzylpenicillin, D-ot-amino-p-trifluoromethylbenzylpenicillin, D-a-aminO-ZA-dibmmobenzylpenicillin, D-a-amino-o-nitrobenzylpenicillin, D-a-amino-mamethylbenzylpenicillin, D-a-amino-m-iodobenzylpenicillin, D-a-amino-o-acetamidobenzylpenicillin, D-a-aminO-Z,6-dichlorobenzylpenicillin, D-a-amino-o-chlorobenzylpenicillin, and D-a-amino-2-chloro-6-fiuorobenzylpenicillin, respectively,

the following penicillins are produced:

Example 19 Potassium 6-{ D-a-allophanamido-3-chlorophenylacetamido}pencillianate.This product is prepared 'by substituting for the ampicillin in the procedure of Example 1 an equimolar weight of 6-[D()-a-amino-3-chlorophenylacetamido]penicillanic acid which is itself prepared by the methods known to the art and also, preferably, by the following procedures:

DL-ot-amino-3-chlorophenylacetic acid.A solution of 250 g. of m-chlorobenzaldehyde in 1.5 l. of 95% ethanol was added in one portion to a stirred solution of 123 g. of sodium cyanide, 515 g. of ammonium carbonate and 1.5 l. of water. The mixture was stirred at 50 for 120 hours. The cooled reaction mixture was acidified to pH 2 with concentrated hydrochloric acid and stirred one hour. The hydantoin was collected by filtration, washed with cold water, and sucked dry on the filter.

A mixture of the crude hydantoin obtained from two runs and 4 l. of 10% sodium hydroxide was refluxed for 18 hours. The solution was carbon treated and neutralized to pH 7 with acetic acid. The solid was col lected by filtration, Washed with water, and dried on the filter. A suspension of the product in 4 l. of water was acidified to pH 2 with concentrated hydrochloric acid. After stirring for 1.5 hours the insoluble material was removed by filtration and the fitrate adjusted to pH 7 with 10% sodium hydroxide. The precipitate was collected by filtration and dried in vacuo at 75 for 18 hours giving 311 g. of DL-a-amino-3-chlorophenylaceti acid; M.P. 266-269 dec.

DL-a-forrnarnido-3-chlorophenylacetic acid.To g. of DL-waminO-B-chlorophenylacetic acid was added 1.33 l. of formic acid. The reaction mixture was warmed to 50 and 483 ml. of acetic anhydride was added dropwise. After storage overnight the DL-a-formamido-3- chlorophenylacetic acid was collected by filtration and washed with water; yield 97 g.

D-(-)-ot-amino-3-chlorophenylacetic acid. a-Formamido-3-chlorophenylacetic acid (721 g.) and one kg. of

dehydroabietylamine were combined in 4 l. of methanol. After storing in the cold for two hours the crystalline salt was collected by filtration. The product was recrystallized from methanol-water; yield 598 g., [a] =225 (c. 0.4 methanol). The salt was slurried in 2 l. of methanol and 2 liters'of saturated sodium bicarbonate solution. The mixture was diluted with 2 l. of water, layered with methyl isobutyl ketone, and stirred vigorously. The aqueous phase was separated and acidified to pH 2 with cone. hydrochloric acid. The acid was collected by filtration and dried. The dried product was combined with 2 l. of 6 N hydrochloric acid and 750 ml. of methanol, the mixture heated for two hours, and filtered. The solution was adjusted to pH 5 with ammonium hydroxide. The solid was collected by filtration and washed with water and acetone giving 112 g. of D-()-a-amino-3- chlorophenylacetic acid; [a] =125 (c. 0.4 1 N HCl). (D-( )-a-amino-3-chlorophenylacetyl chloride hydrochloride.To a stirred suspension of 25 g. of D-()-aamino-3-chlorophenylacetic acid in 375 ml. of methylene chloride at 2 was added 36.5 g. of phosphorus pentachloride. After stirring at 0 to 2 for one and one-half hours the product D-()-m-amino-3-chlorophenylacetyl chloride hydrochloride was collected by filtration, washed with methylene chloride and Skelly Solve B, and dried in vacuo to constant weight; yield 17.0 g.

6-[D-(-)-ot-amino 3 chlorophenylacetamido]penicillanic acid fl-naphthalenesulfonate. A suspension of 32.4 g. (0.150 mole) of 6-aminopencillanic acid in 320 ml. of water was adjusted to pH 6.5 with 20% sodium hydroxide. The resulting clear solution was diluted with droxide and the tetrahydrofuran removed at reduced pres sure on arotary flash evaporator. The aqueous concentrate was filtered. The filtrate was adjusted to pH 2 with 6 N hydrochloric acid and'extracted twice with methyl isobutyl ketone. The aqueous phase was adjusted to pH 4.5 with tricthylamine, layered with ml. of methyl isobutyl ketone and treated with 37.2 ml. of a 43% aqueous solution of fi-naphthalenesulfonic acid. After stirring at to 2 for two hours the crystalline product, 6-[-D-()-a-amino 3 chlorophenylacetamido]penicillanic acid fi-naphthalenesulfonate, was collected by filtration, washed with water, and air dried; yield 11.2 g., M.P. 190-200 dec. with prior softening and darkening above about 175. The infrared spectrum was consistent with 6-[D-(-)-a-amino-3-chlorophenylacetamido] penicillanic acid B-naphthalenesulfonate.

6 [D-()-a-amino-3-chlorophenylacetamido]penicillanic acid.A suspension of 27.3 g. of 6[D-()a amino-3-chlorophenylacetamido]penicillanic acid B-naphthalenesulfonate in 500 ml. of water was adjusted to pH 7.5 with triethylamine and stirred for one hour. The mixture was then slowly adjusted to pH 4.5 with 6 N hydrochloric acid and stirred for two hours at 0. The crystal line product was collected by filtration, washed with water, air dried, and then dried in vacuo over phosphorus pentoxide; yield 12.3 g. A suspension of the product (12 g.) in one 1. of water was adjusted to pH 1.5-1.6 with 6 N hydrochloric acid. The mixture was filtered twice the second time through diatomaceous earth (Super Cel) to remove insoluble material. The clear filtrate was adjusted to pH 4.3 with 20% sodium hydroxide and stirred at 2 for two hours. The crystalline 6-[D-()-oamino 3 chlorophenylacetamido]penicillanic acid was collected by filtration, washed with a small amount of water, and dried in vacuo over phosphorus pentoxide; yield 7.0 g., M.P. 203204 dec., with prior darkening, [a] =-j-26l.9 (c. 0.5 0.1 N HCl).

' Analysis.-Calcd. for C H ClN O S: C, 50.06; H, 4.73; N, 10.92. Found: C, 50.00; H, 4.76; N, 10.5.

This compound is also named 6-[R-a-amino-3-chloro phenylacetamido]penicillanic acid in the nomenclature of Cahn, Ingold and Prelog, Experientia, XII(3), 81-94 (Mar. 15, 1956); all of the D penicillins in this specification are R penicillins in the system of Cahn et al.

Example 20 o ea ll il=i K 1:11

Potassium 6 {D-a-allophanamido-3-fiuorophenylacetamid-o}penicillanate.-This product is prepared by substituting for the ampicillin in the procedure of Example 1 an equimolar weight of 6-[D-()-a-amino-3-fiuoro phenylacetamido]penicillanic acid which is itself prepared by the methods known to the art and also, preferably, by the following procedures:

DL-a-amino-3-fiuorophenylacetic acid.-To a stirred solution of 24.5 g. of sodium cyanide, 29.5 g. of ammonium chloride, 25 ml. of ammonium hydroxide and 100 ml. of water at room temperature was added a solution of 62.0 g. of m-fiuorobenzaldehyde in 200 ml. of methanol. The mixture was stirred at 38 for two hours. The methanol was stripped off at reduced pressure. The residue was extracted with two by 200-ml. portions of ethyl acetate. The combined extracts were washed with water. To the ethyl acetate phase was added dropwise with vigorous stirring 50 ml. of 6 N hydrochloric acid at room temperature. The solution was put under vacuum (water aspirator) and 250 ml. of 6 N hydrochloric acid added dropwise. The mixture was refluxed for 2.5 hours, stirred for 13 hours, and adjusted to pH 4.8 with concentrated ammonium hydroxide While cooling in an ice bath. The aqueous phase was decanted and the gummy precipitate triturated with water and ethyl acetate. The product DL- a-amino-3-fluorophenylacetic acid was collected by filtration and dried in vacuo over phosphorus pentoxide; yield 8.6 g., M.P. 200-203 (sublimation). A second 26 crop of product separated from the filtrates; yield 2.0 g., M.P. 245250 (sublimation).

DL-ot-formamido-3-fluorophenylacetic acid.-A partial solution of 35 g. of DL-oc-amino-3-fluorophenylacetic acid in 356 ml. of 88% formic acid was heated to 50 and 119 ml. of acetic anhydride added dropwise. The mixture was stirred for 17 hours at 50-60 and cooled. The product, DL-u-formamido-3-fluorophenylacetic acid, was collected by filtration and dried in vacuo; yield 38.5 g., M.P. 207209 dec.

D-u-forrnamido-3-fiuorophenylacetic acid-To a solution of 20 g. of DL-u-formamido-3-fluorophenylacetic acid in 4 l. of pH 7 phosphate buffer was added 3.0 g. of hog kidney D-amino acid oxidase (Nutritional Biochemicals Corp.). The mixture was stored at 37 for 19.5 hours, adjusted at pH 5.0 with acetic acid, 5 g. of carbon added, heated to 60 for one-half hour, and filtered. The filtrate was adjusted to pH 2 with 40% phosphoric acid and extracted with ethyl acetate. The ethyl acetate extract was washed with water and stripped to dryness giving 10.0 g. of product; M.P. 190l92, [a] =161.0 (c. 1.0 methanol). The product was again treated with hog kidney D-amino acid oxidase (1 g.) in 500 ml. of pH 7 phosphate butter and the product worked up as above. There was obtained after recrystallization of the product from methanol 5.7 g. of D-u-formamido-3-fiuorophenylacetic acid; [a] =l78.0 (c. 1.0 methanol).

D-a-amino-3-fluorophenylacetic acid.--A suspension of D-u-formamido-3-fluorophenylacetic acid (9.48 g.) in ml. of 6 N hydrochloric acid Was refluxed for one-half hour. The reaction mixture was cooled in an ice bath, filtered, and adjusted to pH 3.8 with concentrated ammonium hydroxide. After stirring for 10 minutes the product, D-u-amino-3-fluorophenylacetic acid, was collected a by filtration and dried in vacuo over phosphorus pentoxide; yield 6.63 g., [a] :110 (c. 1.0 1 N hydrochloric acid), M.P. 249-250".

D-a-(carbobenzoxyamino)-3 -fluorophenylacetic acid.- To D-a-amino-3-fluorophenylacetic acid (4.9 g.) partially dissolved in 300 ml. of water at pH 10 (10% sodium hydroxide used) at 0 was added 11.2 g. of carbobenzoxy chloride. The vigorously stirred mixture was maintained at pH 10 by the addition of 10% sodium hydroxide. A white gum formed. After one hour the reaction mixture was extracted once with ether. The aqueous phase was layered with ethyl acetate and acidified to pH 3 with 6 N hydrochloric acid. Three additional extractions with ethyl acetate were made. The combined ethyl acetate extracts were washed with water, dried with magnesium sulfate, the solvent stripped off at reduced pressure, the resulting residual oil solidified with Skelly Solve B, collected by filtration, and dried in vacuo over phosphorus pentoxide; yield 4.3 g. of D-a-(carbobenzoxyamino)-3-fluorophenylacetic acid, M.P. 117119, [u] =8O (c. 1.0 methanol).

6-(D-a-amino 3 fiuorophenylacetamido)penicillanic acid.Ethyl chloroformate (1.53 g.) was added to a solution of 4.2 g. of D-u-(carbobenzoxyamino)-3-fluorophenylacetic acid and 1.94 ml. of triethylamine in 70 ml. of tetrahydrofuran at 5. After stirring for 10 minutes a solution of 3.03 g. of 6-aminopenicillanic acid and 1.94 ml. of triethylamine in 70 ml. of 50% aqueous tetrahydrofuran was added all at once. The mixture was stirred for one-half hour at 0 and then the ice bath was removed for one hour. The reaction mixture was diluted with 250 ml. of Water and the tetrahydrofuran distilled off at reduced pressure. To the residue was added 350 ml. of ethyl acetate and 7 ml. of 42% phophoric acid. Two additional extractions with 250-ml. portions of ethyl acetate were made. The combined ethyl acetate extracts were washed with water, dried with magnesium sulfate, and the solvent distilled off at a reduced pressure. The residue was solidified with Skelly Solve B, collected by filtra tion, and dried in vacuo over phosphorus pentoxide giving 4.8 g. of 6-[D-a-(carbobenzoxyamino)-3-fluorophenylacetamido]-penicillanic acid.

The carbobenzoxy penicillin was dissolved in 50 ml. of 3% sodium bicarbonate plus 50 ml. of dioxane and hydrogenated in the presence of 4.8 g. of 30% palladium on diatomaceous earth for 25 minutes on a low pressure Paar hydrogenation apparatus. Phosphoric acid (42%) was added to the hydrogenation mixture to coagulate the catalyst. The mixture was filtered, methyl isobutyl ketone was added to the filtrate, and the mixture again filtered removing all catalyst. The aqueous phase was separated, adjusted to pH 4.5 with 10% sodium hydroxide, and the dioxane distilled off at reduced pressure. The concentrate was readjusted to pH 4.5, 2-propano1 added, and concentrated giving a gelatinous precipitate which was collected by filtration and washed with ether; yield 13.8 g. The crude product was stirred with ml. of water for 30 minutes, collected by filtration, and dried in vacuo over phosphorus pentoxide; yield 700 mg. The infrared and nuclear magnetic resonance spectra were consistent with 6-(D-a-amino-3-fluorophenylacetamido)penicillanic acid.

I claim:

1. The compounds having the formula R is hydrogen or (lower) alkyl;

R R and R each is hydrogen, nitro, (lower)alkylamino, di(lower)alkylamino, (lower) alkanoylamino, amino, hydroxy, (lower)alkanoyloxy, (lower) alkyl comprising straight and branched chain saturated A aliphatic groups having from 1 to 6 carbon atoms inclusive, (lower)alkoxy, sulfamyl, chloro, iodo, bromo, fluoro or trifluoromethyl; and their nontoxic, pharamceutically acceptable salts.

2. The compounds of claim 1 wherein R R and R are each hydrogen, hydroxy or amino.

3. The compounds of claim 1 wherein R is hydrogen or methyl, R is hydrogen, hydroxy or amino and R and R are hydrogen.

4. The compounds of claim 1 wherein R R and R are each hydrogen.

5. The compounds of claim 1 wherein R, R R and R are each hydrogen.

6. The compounds of claim 1 wherein R R and R are each hydrogen and R is (lower)alkyl.

7. The compounds of claim 1 wherein R R and R are each hydrogen and R is methyl.

8. The compound of claim 1 known as 6-[D-a-(allophanamido) phenylacetamido]penicillanic acid and its nontoxic, pharmaceutically acceptable salts.

9. The compound of claim 1 known as 6-[D-a-(5-methylallophanamido)-phenylacetamido]penicillanic acid and its nontoxic, pharmaceutically acceptable salts.

10. The compound of claim 1 known as 6-[D-rx-(allophanamido)-2-thienylacetamido]penicillanic acid and its nontoxic, pharmaceutically acceptable salts.

11. The compound of claim 1 known as G-[D-a-(S- methylallophanamido) 2 thienylacetamido]penicillanic acid and its nontoxic, pharmaceutically acceptable salts.

12. The compound of claim 1 known as G-[D-a-(allophanamido)-3-thienylacetamido]penicillanic acid and its nontoxic, pharmaceutically acceptable salts.

13. The compound of claim 1 known as 6-[D-a(5- methylallophanamido) 3 thienylacetamido] penicillanic acid and its nontoxic, pharmaceutically acceptable salts.

14. 6-[D-ot-(allophanamido) phenylacetamido] penicillanic acid.

15. 6-[D-a-(allophanamido)-2-thienylacetamido] penicillanic acid.

16. 6-[D-a-(allophanamido-3-thienylacetamido] penicillanic acid.

17. Sodium 6-[D-ot-(allophanamido)phenylacetamido]- penicillanate.

18. Sodium 6-[D-u-(allophanamido)-2-thienylacetamido]-penicil1anate.

19. Sodium 6-[D-u-(allophanamido)-3-thienylacetamidoj-penicillanate.

20. Potassium 6-[D-a-(allophanamido)phenylacetamido]-penicillanate.

21. Potassium 6-[D-a-(allophanamido)-2-thienylacetamido]-penicillanate.

22. Potassium 6-[D-u-(allophanamido)-3-thienylacetamido] -penicillinate.

References Cited UNITED STATES PATENTS 3,308,023 3/1967 Russell 260239.1

NICHOLAS S. RIZZO, Primary Examiner U.S. Cl. X.R. 42427l 

